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Toxicological information

Genetic toxicity: in vivo

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Administrative data

Endpoint:
in vivo mammalian germ cell study: gene mutation
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2014-2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was conducted according to test guidelines and in accordance with GLP.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
other: OECD Technical Guideline 488
Deviations:
yes
Remarks:
Archive samples taken after completion dosing, cage side observations not done on one date, sampling location for one sample for one sample, and no necropsy data or death details for one control anima
Principles of method if other than guideline:
not applicable
GLP compliance:
yes
Type of assay:
transgenic rodent mutagenicity assay

Test material

Constituent 1
Reference substance name:
Diethylenetriamine (DETA)
IUPAC Name:
Diethylenetriamine (DETA)
Constituent 2
Chemical structure
Reference substance name:
2,2'-iminodi(ethylamine)
EC Number:
203-865-4
EC Name:
2,2'-iminodi(ethylamine)
Cas Number:
111-40-0
Molecular formula:
C4H13N3
IUPAC Name:
N-(2-aminoethyl)ethane-1,2-diamine
Test material form:
other: Clear yellow liquid
Details on test material:
- Name of test material (as cited in study report): Diethylenetriamine (DETA)
- Physical state: Clear yellow liquid
- Analytical purity: 98.9%
- Lot/batch No.: F286E6QL79
- Expiration date of the lot/batch: 31 July 2016
- Storage condition of test material: Room temperature, protected from light, with nitrogen

Test animals

Species:
mouse
Strain:
C57BL
Sex:
male
Details on test animals or test system and environmental conditions:
TEST ANIMALS Group 1 (C57BL/6 (wildtype) strain)
- Source: Taconic Farms Inc., Germantown, NY
- Age at study initiation: 10 weeks
- Weight at study initiation: 22.8 to 26.4 grams
- Assigned to test groups randomly: Yes
- Housing: Animals were housed in an Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC)-accredited facility following the specifications recommended in the most current version of The Guide for the Care and Use of Laboratory Animals (National Academy Press, Washington, D.C.).
- Diet (e.g. ad libitum): All animals received Harlan TEKLAD Global Diet #2018CM (Certified 18% Protein Rodent Diet, Harlan TEKLAD, Madison, Wisconsin), in
stainless steel rodent feeders, ad libitum from arrival until termination of the 5-Day and 28-Day studies.
- Water (e.g. ad libitum): Drinking water from an automatic watering system was provided ad libitum from arrival until termination of the 5-Day and 28-Day studies.
- Acclimation period: The animals were acclimated for 6 days prior to the first test and/or control article administration in both the 5-Day and 28-Day studies.

TEST ANIMALS Group 2 (Big Blue® C57BL/6)
- Source: Taconic Farms Inc., Germantown, NY
- Age at study initiation: 11 weeks
- Weight at study initiation: 18.0 to 24.0 grams
- Assigned to test groups randomly: Yes
- Housing: Animals were housed in an Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC)-accredited facility following the specifications recommended in the most current version of The Guide for the Care and Use of Laboratory Animals (National Academy Press, Washington, D.C.).
- Diet (e.g. ad libitum): All animals received Harlan TEKLAD Global Diet #2018CM (Certified 18% Protein Rodent Diet, Harlan TEKLAD, Madison, Wisconsin), in
stainless steel rodent feeders, ad libitum from arrival until termination of the 5-Day and 28-Day studies.
- Water (e.g. ad libitum): Drinking water from an automatic watering system was provided ad libitum from arrival until termination of the 5-Day and 28-Day studies.
- Acclimation period: The animals were acclimated for 6 days prior to the first test and/or control article administration in both the 5-Day and 28-Day studies.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 69 - 75°F
- Humidity (%): 30 - 70%
- Air changes (per hr): At least 10 changes of fresh HEPA-filtered air every hour
- Photoperiod (hrs dark / hrs light): 12-hour light/12-hour dark cycle

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: None
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: A dose volume of 10 mL/kg body weight, for up to 5 and/or 28 consecutive days. Individual dose volumes were calculated based on the animal’s most recently recorded body weight.

DIET PREPARATION
Formulations were mixed for at least 30 minutes prior to dosing and throughout dosing; formulations were used within 2 hours of removal from storage and were capped when not in use.
Duration of treatment / exposure:
5 and 28 days
Frequency of treatment:
5 day range finding study: Administered once daily by oral gavage for up to 5 consecutive days.

28 day: All animals in Groups 1 through 4 were dosed once daily by oral gavage for 28 consecutive days beginning on Day 1; positive control animals (Group 5) were dosed once daily by oral gavage within two hours after preparation on Days 1, 2, and 3.
Post exposure period:
All animals were observed twice daily, at least 6 hours apart, for moribundity and mortality. Cage side observations were performed daily, on days of dosing, within 2 hours after the last animal was dosed in each group, except as noted (see Deviations from the Protocol). Positive control
animals had cage side observations only on Days 1, 2, and 3. Detailed hands-on observations were performed on Day 1 and weekly thereafter.
Doses / concentrations
Remarks:
Doses / Concentrations:
10 mL/kg
Basis:
nominal conc.
No. of animals per sex per dose:
42 males
Control animals:
yes
Positive control(s):
none; no data; 2-acetylaminofluorene;N-ethyl-N-nitrosourea (ENU) in Buffer Solution pH 6.00
- Route of administration: oral
- Doses / concentrations: One vial containing 1 gram of the ENU-ISOPAC® was removed from storage (-10 to -30°C) and allowed to warm to room temperature. The buffer solution was measure by volume into a syringe.

Examinations

Tissues and cell types examined:
Bone marrow, liver and stomach tissues samples from 5 animals/group were processed for DNA isolation. Testes/cauda tissues samples were not processed, since no increase in mutations was detected in any of the tissues initially examined. Liver and bone marrow were extracted following BioReliance SOP PROC-BREL_US-OP-037268, which was based on methods described for Agilent product RecoverEase (Agilent, 2009a) for somatic tissues. DNA extraction of stomach used modified methods similar to the Agilent method except that one half of the glandular stomach was scraped in Phosphate Buffered Saline (PBS), the outer layer of the stomach discarded, the stomach lining in PBS centrifuged, the pelleted cells homogenized and nuclei pelleted as per the Agilent method. From this point the pelleted nuclei were digested, phenol/chloroform extracted, precipitated with ethanol and dissolved in TE buffer (as described in Young et. al, 2015). Isolated DNA in all cases was stored at 2-8°C.
Details of tissue and slide preparation:
Isolated DNA were processed using Agilent Transpack packaging extract, in order to isolate the recoverable lambda shuttle DNA vectors from the genomic DNA and to package the lambda shuttle vector DNA into empty phage capsids creating infectious phage particles. Methods followed BioReliance SOP PROC-BREL_USOP-035769 that was based on Agilent instruction manual titled “λ Select-cII Mutation Detection System for Big Blue Rodents” (Agilent, 2009b) and Agilent instruction manual titled “Transpack Packaging Extract for Lambda Transgenic Shuttle Vector Recovery” (Agilent 2009c) Frozen stocks of E. coli strain G1250, provided and characterized by Agilent as part of λ Select-cII Mutation Detection System for Big Blue Rodents, were used toprepare master bacterial plates. Several colonies were picked from master plates and used to prepare overnight suspension cultures for plating of phage for plaque formation. Packaged phage was adsorbed onto E. coli G1250 suspension cultures for 30 minutes, molten top agar added and the cells plated onto bottom agar plates. Plates with diluted phage were incubated at 37° ± 1.0°C overnight for calculation of phage titer. Plates with undiluted phage were incubated at 24°C ± 0.5°C for 42 to 48 hours for determination of the number of mutants. After incubation, plates were
scored visually for number of plaques per plate.
Evaluation criteria:
The individual animal is considered the experimental unit. The mutant frequency (MF) was calculated (number of mutant phage / number of total phage screened) for each tissue analyzed from each animal. Since this ratio is extremely small and may not be normally distributed, a log10 transformation of the MF data was performed. The statistical analysis of MF was conducted in two parts. Initially, the positive control group was compared to the vehicle control. In the second part of the analysis, all the groups (except the positive control) were compared to the vehicle control. In both cases, log10 transformed MF data from the vehicle control and treated groups were evaluated using a 1-Way Analysis of Variance (ANOVA). The
suitability of using the parametric ANOVA was confirmed by testing parameters of the log10 transformed MF data for normality and equal variance. If the data are normally distributed and exhibit equal variance, the parametric ANOVA analysis would be used. Otherwise, if either test fails, a non-parametric analysis of variance would be used.
Statistics:
Dunnett’s test was conducted on body weight, body weight changes, and organ weight data. The use of Fisher's Exact Test to compare frequency data (incidence of mortality and clinical observations) was not deemed necessary by the Study Director. All required statistics were based on a significance value of p < 0.05.

Results and discussion

Test results
Sex:
male
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Treatment with DETA did not cause statistically elevated mutant frequency at the cII gene in bone marrow, liver and stomach of Big Blue mice. Group mean cII mutant frequencies of the positive control animals were statistically elevated (p = 0.002 for liver and p < 0.001 for bone marrow and stomach) over the respective vehicle control values, and were consistent with historical experience. DETA was evaluated as negative for the induction of cII mutants in liver, bone marrow and stomach in male Big Blue C57BL/6 mice under the conditions of testing. The study design and results obtained met protocol-specified assay acceptance criteria and were consistent with the study requirements of OECD Test Guideline 488 for transgenic rodent mutation assays.

Any other information on results incl. tables

A. Statistical Analysis of Liver Mutant Frequency Data

1. Positive control (Group 5) vs. Vehicle control (Group 1): 1-Way ANOVA revealed that the mean mutant frequency of the ENU-treated group was significantly elevated over the mean mutant frequency of the vehicle control group (p = 0.002). The normality test performed on the

residuals (p > 0.100) and the equal variance test (p = 0.718) confirmed that

the ANOVA criteria were met, therefore the analysis was considered

adequate.

2. Test Article Groups (Groups 2, 3 and 4) vs. Vehicle control (Group 1):

1-Way ANOVA revealed that the mean mutant frequencies of the

Diethylenetriamine-treated groups were not significantly different than the

mean mutant frequency of the vehicle control group (p = 0.099). The

normality test performed on the residuals (p > 0.100) and the equal variance

test (p = 0.319) confirmed that the ANOVA criteria were met, therefore the

analysis was considered adequate.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative
Treatment with DETA did not cause statistically elevated mutant frequency at the cII gene in liver, bone marrow, and stomach of Big Blue mice. The positive control treatment with ENU produced statistically significant increases in mutant frequencies for all tissues tested, demonstrating the utility of the test system to detect and quantify induced mutants following exposure to a known direct acting mutagen. The study design and results obtained were consistent with the study requirements of OECD Test Guideline 488 for transgenic rodent mutation assays.
Executive summary:

 

The purpose of this study was to determine the effect of Diethylenetriamine (DETA) on mutation frequency at the cII gene in liver, bone marrow, and stomach from male transgenic homozygous C57BL/6 Big Blue® mice. The Big Blue® Assay is a Transgenic Rodent (TGR) mutation assay, described in OECD Technical Guideline 488 (OECD, 2013).

 

The range-finding portion of the study consisted of four groups of three male mice/group. Mice were 10 weeks old at dosing initiation. Vehicle control animals (Group 1) received the vehicle (de-ionized water) and test article-treated animals received DETA formulated in the vehicle at doses of 400, 700 and 1000 mg/kg/day (Group 2, 3 and 4, respectively). Animals in Groups 1-4 were dosed by oral gavage for up to 5 consecutive days, at a dose volume of 10 mL/kg. Animals were sacrificed by CO2 overdose on Day 5 after completion of blood collection (for bioanalysis).

 

The transgenic mutation portion of the study consisted of five groups of six male mice/group. Mice were 11 weeks old at dosing initiation. Vehicle control animals (Group 1) received the vehicle (de-ionized water) and test article- treated animals received DETA formulated in the vehicle at doses of 50, 150 and 400 mg/kg/day (Group 2, 3 and 4, respectively). Animals in Groups 1-4 were dosed once daily by oral gavage for 28 consecutive days, at a dose volume of 10 mL/kg. The positive control animals (Group 5) were dosed via oral gavage on Days 1, 2 and 3 with 40 mg/kg/day of N-ethyl-N-nitrosourea (ENU) formulated in phosphate buffer solution, pH 6.00. ENU was also administered at a dose volume of 10 mL/kg. Animals were sacrificed by CO2 overdose on Day 31 and tissues were collected, weighed (except for bone marrow), flash frozen and stored at -60°C or less for later mutant analysis. Protocol specified tissues were processed for DNA isolation and analysis of cII mutants following BioReliance SOP’s.

 

End points evaluated during the study included mortality, clinical signs, body weights (and body weight changes), organ weights, bioanalytical and mutant frequency data.

 

In the 5-Day range finding study, all animals treated at 1000 mg/kg/day were terminated early due to moribundity. Stomach discoloration was noted in 2/3 animals treated at 700 mg/kg/day and is believed to be related to treatment with DETA. There were no other toxicologically relevant findings in the DETA treated groups. Positive exposure of DETA to the animals was demonstrated in all analyzed DETA treated groups.

 

In the transgenic mutation, 28-Day study, all animals survived until scheduled sacrifice on Day 31. There were no other toxicologically relevant findings in any of the DETA treated groups. Clinical observations, changes in body weights and organ weights, and gross necropsy findings in ENU treated groups were consistent with the toxicity of the positive control.

 

Vehicle control (Group 1) samples demonstrated reproducible background cII mutant frequencies (MF) in the liver, bone marrow, and the stomach, generally consistent with past experience. Group cII mutant frequencies in DETA-treated animals were 46.5 ± 13.9, 44.9 ± 9.9 and 47.6 ± 6.4 x 10-6 in liver, 39.0 ± 14.8, 38.8 ± 21.4 and 41.1 ± 6.6 x 10-6 in bone marrow, and 44.9 ± 7.1, 50.8 ± 19.0 and 46.8 ± 14.1 x 10-6 in the stomach (Groups 2, 3 and 4, respectively). Group means MF for all dose levels for the three tissues were within historical background for the respective tissue and were not statistically significant when compared to the vehicle control. Group mean cII mutant frequencies of the positive control animals were statistically elevated (p= 0.002 for liver and p < 0.001 for bone marrow and stomach) over the respective vehicle control values, and were consistent with historical experience.  

 

Treatment with DETA once daily by oral gavage for 28 days, at dose levels of 50, 150, and 400 mg/kg/day, did not cause statistically elevated mutant frequency at the cII gene in liver, bone marrow, and stomach of Big Blue mice. The positive control treatment with ENU produced statistically significant increases in mutant frequencies for all tissues tested, demonstrating the utility of the test system to detect and quantify induced mutants following exposure to a known direct acting mutagen. There were no toxicological effects observed that may have altered the outcome of this study.

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